Somatic Cell Reprogramming And Generation Of Induced Multipotent Endodermal Stem Cells

The liver is the organ that plays an important role in metabolism and has a number offunctions in the body, including glycogen storage, decomposition of red blood cells,plasma protein synthesis, hormone production, and detoxification. End-stage liverdisease caused by a variety of reasons is a major hazard to human health[1], which ishighly prevalent in China. So far, the only effective treatment for end-stage liver diseaseis liver transplantation. But due to the shortage of donors, long-term use ofimmunosuppressant after the transplantation together with related economic problemsand possible complications, as well as the risks of transplantation, its application hasbeen seriously impeded[2]. With the development of stem cell technology, cell therapyhas become a new option for end-stage liver disease treatment[3]. And how to get clinicalsafe, adequate, obtainable source of seed cells is the most important issue that needs tobe addressed. Several kinds of cells have been employed for clinical treatment, includingthe mesenchymal stem cells and fetal or carcasses liver cells. However, the ethical issuesand the graft rejection caused by them have limited their application in liver diseasetherapy.Embryonic stem cell (ESC) is a self-renewal and totipotent stem cell, whichtheoretically can be differentiated into functional cells of all types in the body. Therefore,it has a great application potential in therapies of many diseases, drug screening anddisease modeling. However, as ESC is derived from the inner cell mass (ICM) of adeveloping embroy, the ethical issues greatly hindered its application. In addition, theimmunological rejection caused by cell transplantation and the risk of tumorigenicityalso restrict its application.Induced pluripotent stem cell (iPSC) is an ESC-like pluripotent cell generated fromsomatic cell through reprogramming technology. Not only does it have thedifferentiation potential as ESC, but also it avoids the ethical issues related to the ESCapplication. Besides, as iPSC could be generated from patients' somatic cells, it`s moreconducive to the individualized treatments by patient-or disease-specific iPSC. Since 2007, numerous studies have reported iPSC generated from almost all kinds of adultcells through the classic reprogramming method established by Yamanaka[5][6]andfurther studies have confirmed that iPSC can differentiate into hepatocytes[7],cardiomyocytes[8], blood cells[9]and other functional cells. However, the risk oftumorigenicity during transplantation is still a problem that hindered its applications.Additionally, by lineage reprogramming, a few studies have demonstrated that anadult cell type can be directly converted to another adult cell type. For cell-replacementtherapies, the idea of lineage reprogramming is fascinating because of its potential torapidly generate a variety of therapeutically important and immunologically matchedcell types directly from one's own easily accessible tissues.This thesis is mainly divided into two parts. In the first part, we used the classicsomatic cell reprogramming method to generate iPSC fom the patient's skin fibroblasts(human foreskin fibroblast, HFF) cells, and further confirmed the in vitro differentiationpotential of it. In the second part, epithelial stem/progenitor cells were isolated fromgastrointestinal (GI) tract which is developmentally homologous with the liver. Then wereprogrammed them into induced endodermal mutipotent stem cells (iEMSC) whichwere morphologically and characteristically similar to definitive endoderm, in aclinically safe and virus-independent way. And finally, we generated functionalhepatocytes from iEMSC.PartⅠGeneration of iPSC from somatic cells by pluripotency related transcription-al factorsIn2006, for the first time, Yamanaka Laboratory generated iPSC by transduction ofpluripotent cell-specific transcriptional factors Oct4, Klf4, c-Myc, and Sox2into HFF.Since then, numerous studies have established iPSCs from almost all types of terminallydifferentiated adult cells by similar methods, and demonstrated that iPSC can bedifferentiated into functional cardiomyocytes, neurons, hematopoietic cells, hepatocytes,pancreatic cells, etc.Here, we demonstrated the generation of iPSC from HFF cells with adenoviral andretroviral vectors expressing Oct3/4, Sox2, Klf4, and c-Myc. iPSC resemble ESC closelyin terms of morphology, proliferation properties, surface antigens and gene expression.We analysed the expression of the endogenous loci and transgenes, and found thatendogenous expression of Oct4, Sox2, c-Myc and Klf4was comparable to human EScells, and accompanied by silencing of exogenous genes, confirming that the iPS cells were fully reprogrammed.In order to verify the in vitro differentiation capacity of the iPSC, we co-culturediPSC and ESC with the Wnt3a gene-modified OP9cell line (Wnt3a-OP9) forhematopoietic differentiation. We found that the number of both Flk1+and CD41+cellsgenerated from both co-cultured iPSC and ESC increases significantly. The percentageof Flk1+cells were47.11±2.61%of miPSC-OP9and50.04±1.71%of miPSC-Wnt3a-OP9cells,22.08±1.72%of mESC-OP9and31.90±1.25%of mESC-Wnt3a-OP9on day3.75. The rate of CD41~+cells were1.53±0.29%of miPSC-OP9and4.53±0.24%ofmiPSC-Wnt3a-OP9cells, and1.29±0.19%of mESC-OP9and2.76%±0.13ofmESC-Wnt3a-OP9on day5. Taken together, in this section of our studywe confirmedthe in vitro hematopoietic differentiation capacity of the iPSC. Besides, we proved thatWnt3a was involved in the early stages of hematopoietic differentiation of both ESC andiPSC.By co-culturing iPSC derived EBs with mouse tooth germ mesenchymal cells, andthen transplanting them in the mouse renal cysts for4weeks, tooth bud formation couldbe observed. RT-PCR results showed a significate increase in the expression of dentalepithelial cells specific genes Msx1, Lhx7, Pax9, and Runx2, indicating that the iPSCcould differentiate into dental epithelial cells.Together, our study in this part established iPSC and confirmed its pluripotency,which provided a research platform for generation patient-/diease-specific seed cells forcell therapy of end-stage liver disease.PartⅡGeneration of iEMSC from GI tract epithelial stem/progenitor cells with acombination of small moleculesAlthough iPSC retains the advantages of the ESC, and avoids the ethical controversyand immunological rejection, its clinical application is still greatly hindered by itschromosomal abnormalities caused by genetic modification, the introduction of virusduring the reprogramming process, the risk of tumorigenicity, and the low efficiency ofiPSC generation. However, these limitations may be circumvented by using directlineage reprogramming which is fast, efficient and developmentally restricted.In this part of the study, we reprogrammed epithelial stem/progenitor cells isolatedfrom gastrointestinal (GI) tract into induced endodermal mutipotent stem cell (iEMSC)with only chemical compounds. As iEMSC was expandable and able to differentiate into multiple lineages, they might serve as the desired seed cells for cell therapy in end-stageliver disease.Histochemistry analysis showed that CK19, CXCR4, EpCAM, FoxA2, Sox9, Lgr5positive epithelial stem/progenitor cells existed in the sites of gastric glands, duodenalsubmucosal glands and colon crypt of samples obtained from clinical operations. Theprimary epithelial stem/progenitor cells with limited proliferation potential were isolatedfrom fresh samples according to the protocol of primary epithelial cell isolation. Andthey were efficiently reprogrammed into iEMSC with a combination of small molecules.iEMSC resembled DE in terms of morphology and gene expression profile.Immnostainning and qPCR results showed that iEMSC expressed Sox17, FoxA2. Andthe promoters of Sox17and FoxA2were in hypomelthylation state in iEMSC. Inaddition, iEMSC was stably expandable and its doubling time was1.768day. And itmaintained a normal karyotype. Further in vitro differentiation showed that iEMSCcould differentiate into hepatocytes, pancreatic cells, intestinal cells and bile duct cells.In summary, by selecting epithelial stem/progenitor cells with differentiationcapacity but limited proliferation potential as the starting cells, we generated iEMSCwith only chemical compounds in a safe and efficient way, thus providing a clinicallysafe and controllable source of seed cells for cell therapy of end-stage liver disease in thefuture.